Ivor J. Lim

Investigation of the influence of keloid-derived keratinocytes on fibroblast growth and proliferation in vitro

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber. Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.

Complex epithelial–mesenchymal interactions modulate transforming growth factor-β expression in keloid-derived cells

Keloids are proliferative dermal growths representing a pathologic wound healing response. We have previously demonstrated that coculture of fibroblasts derived from either keloid or normal skin have an elevated proliferation rate when cocultured with keloid‐derived keratinocytes vs. normal keratinocytes. In these studies, we examined the contribution of transforming growth factor‐β (TGF‐β) to this phenomenon using a two‐chamber coculture system. Fibroblast proliferation in coculture was slower with the addition of a pan‐TGF‐β neutralizing antibody. Keloid keratinocytes in coculture expressed more TGF‐β1, ‐β3, and TGF‐β receptor 1 than normal keratinocytes. Keloid fibroblasts cocultured with keloid keratinocytes expressed more mRNA for TGF‐β1, ‐β2, TGF‐β receptor 1, and Smad2. Keloid fibroblasts also produced more type I collagen, connective tissue growth factor, and insulin‐like growth factor‐II/mannose‐6‐phosphate receptor when cocultured with keloid keratinocytes vs. normal keratinocytes. Levels of total and activated TGF‐β activity increased when fibroblasts were cocultured with keratinocytes, correlating with the changes in transcriptional activity of TGF‐β. In conclusion, we find a complex paracrine interaction regulates TGF‐β mRNA expression and activation between keratinocytes and fibroblasts. These data suggest that keloid pathogenesis may result from both an increased TGF‐β production and activation by the keloid keratinocyte, and elevated TGF‐β expression, utilization, and signaling in keloid fibroblasts.

Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte‐fibroblast coculture contributes to keloid pathogenesis

Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial‐mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial‐mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast‐single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial‐mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions. J. Cell. Physiol. 208: 336–343, 2006.

Smad3 signalling plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions

Smad signalling plays important roles in developmental and cancer biology as well as in fibropathogenesis. Its role in keloid biology is not known. Epithelial–mesenchymal interactions, originally described in normal skin, have recently been established to play a significant role in keloid pathogenesis, and demonstrate the important influence of keratinocyte paracrine factor signalling on fibroblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. Normal fibroblasts (NF) and keloid fibroblasts (KF) were co‐cultured in serum‐free medium with normal keratinocytes (NK) or keloid keratinocytes (KK) for 5 days, after which fibroblast cell lysates were subjected to western blot and immunoprecipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild‐type (wt), Smad2‐null (Smad2−/−) and Smad3‐null (Smad3−/−) mouse embryonic fibroblasts (MEF) were assayed for cell proliferation and collagen production after serum‐free co‐culture with KK or exposure to conditioned media collected from serum‐free KK/KF co‐culture. Compared to normal skin, keloids expressed high basal levels of TGFβR1 and TGFβR2, Smad2, 3 and 4 and phospho‐Smad2. Upregulation of TGFβR1 and TGFβR2, Smad3 and p‐Smad2 was observed in KF co‐cultured with KK, together with enhanced Smad3 phosphorylation and Smad2/3/4 binding complex production. When MEF‐wt, MEF‐Smad2−/− or MEF‐Smad3−/− were co‐cultured with KK or exposed to KK/KF co‐culture conditioned media, enhanced proliferation and collagen production were seen in MEF‐wt and MEF‐Smad2−/− but not in MEF‐Smad3−/− cells. The activation of Smad signalling, importantly that of Smad3, appears to be one facet of the complex epithelial–mesenchymal interactions in keloid pathogenesis, resulting in active KF proliferation and collagen‐ECM production in co‐culture with KK. This finding suggests the suppression of Smad signalling as a novel approach in keloid therapy. Copyright

Suppression of transforming growth factor beta/smad signaling in keloid-derived fibroblasts by quercetin: implications for the treatment of excessive scars.

BACKGROUND Keloids are characterized by abnormal proliferation and overproduction of extracellular matrix. Quercetin, a dietary compound, has strong antioxidant and anticancer properties. Previous studies by the authors have shown that quercetin inhibits fibroblast proliferation, collagen production, and contraction of keloid and hypertrophic scar-derived fibroblasts. Quercetin also blocks the signal transduction of insulin-like growth factor-1 in keloid fibroblasts. This study assessed the effects of quercetin on the transforming growth factor (TGF)-beta/Smad-signaling pathway in keloid-derived fibroblasts, which may be an important biologic mechanism of this proliferative scarring. METHODS Keloid fibroblasts were isolated from keloid tissue specimens. Cells were treated with quercetin at different concentrations, then harvested, and subjected to immunoblotting analysis. RESULTS Quercetin significantly inhibited the expression of TGF-beta receptors 1 and 2 in keloid fibroblasts at three concentrations (low, medium, and high). Quercetin also strongly suppressed the basal expression of Smad2, Smad3, and Smad4 as well as the phosphorylation of Smad2 and Smad3 and the formation of the Smad2-Smad3-Smad4 complex. CONCLUSIONS Taken together, these data suggest that quercetin effectively blocks the TGF-beta/Smad-signaling pathway in keloid fibroblasts. These data indicate that quercetin-based therapies for keloids should be investigated further.

From scarless fetal wounds to keloids: Molecular studies in wound healing

Surgical researchers were among the first to describe the different phases of wound healing and the events in tissue repair and regeneration that were taking place during each phase. The understanding of these events has been significantly enhanced in recent years by modern techniques in molecular and cellular biology. In this article, we discuss new findings in scarless fetal repair, angiogenesis in wound healing, and keloid pathogenesis. This serves to highlight the advances that have been made and also how much remains to be understood. (WOUND REP REG 2003;11:411–418)

Synchronous Activation of ERK and Phosphatidylinositol 3-Kinase Pathways Is Required for Collagen and Extracellular Matrix Production in Keloids

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2–5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I–III, laminin β2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I–III production and significantly decreased laminin β2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I–III and laminin β2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.

mTOR as a potential therapeutic target for treatment of keloids and excessive scars

Abstract:  Keloid is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix (ECM) components such as collagen, glycoproteins and fibronectin. The mammalian target of rapamycin (mTOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. Published reports have shown mTOR as regulator of collagen expression and its inhibition induces a decrease in ECM deposition. Our aim was to investigate the role of mTOR in keloid pathogenesis and investigate the effect of rapamycin on proliferating cell nuclear antigen (PCNA), cyclin D1, collagen, fibronectin and alpha‐smooth muscle actin (α‐SMA) expression in normal fibroblasts (NF) and keloid fibroblasts (KF). Tissue extracts obtained from keloid scar demonstrated elevated expression of mTOR, p70KDa S6 kinase (p70S6K) and their activated forms, suggesting an activated state in keloid scars. Serum stimulation highlighted the heightened responsiveness of KF to mitogens and the importance of mTOR and p70S6K during early phase of wound healing. Application of rapamycin to monoculture NF and KF, dose‐ and time‐dependently downregulates the expression of cytoplasmic PCNA, cyclin D1, fibronectin, collagen and α‐SMA, demonstrating the anti‐proliferative effect and therapeutic potential of rapamycin in the treatment of keloid scars. The inhibitory effect of rapamycin was found to be reversible following recovery in the expression of proteins following the removal of rapamycin from the culture media. These results demonstrate the important role of mTOR in the regulation of cell cycle and the expression of ECM proteins: fibronectin, collagen and α‐SMA.

Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation

Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF‐β isoforms and collagen Iα, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF‐β1, TGF‐β2, and collagen Iα after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin β1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro‐fibrotic growth factors. This may be one molecular mechanism for the development of keloids. J. Cell. Physiol. 206: 510–517, 2006.

Epithelial–mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor expression and secretion†

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis during the wound healing process. As epithelial–mesenchymal interactions have been shown to regulate a plethora of genes in wound healing, we hypothesized that these interactions might have a role in modulating VEGF expression and angiogenesis. A two chamber co‐culture model was used, wherein normal and keloid keratinocytes and fibroblasts were physically separated by membrane inserts while allowing cytokine diffusion. Cell lysates obtained from keratinocytes co‐cultured with fibroblasts demonstrated increased expression of VEGF. An enzyme‐linked immunosorbent assay (ELISA) showed significant increase in VEGF expression in co‐culture conditioned media compared with controls. Additionally, the conditioned medium from keloid keratinocyte and fibroblast co‐cultures increased proliferation and formation of complex three‐dimensional capillary‐like structures in human umbilical vein endothelial cells, emphasising the importance of epithelial–mesenchymal interactions in the angiogenic process. Immunostaining of keloid tissue localized VEGF in the basal layer of the epidermis and also demonstrated higher blood vessel density than normal skin. Keloid tissue extract also demonstrated increased expression of VEGF compared with normal skin. It is likely that epidermal VEGF exerts significant paracrine control over the dynamics and expression profile of underlying dermal fibroblasts. Addition of the inhibitors WP631, mitoxantrone, and Rapamycin to keloid keratinocyte and fibroblast co‐cultures, downregulated secreted VEGF expression in a dose‐dependent manner, suggesting therapeutic potential for these compounds in the treatment of keloid scars. Copyright